The development of rapid molecular diagnostic tests for human infections is the most highly rated priority of the World Health Organization for health improvement of the world population (Daar et al., 2002, Nat. Genet., 32:229-232). Severe blood infections are an important cause of morbidity and death in hospitalized patients worldwide and one of the most important challenges in critical care. For example, recent estimates of sepsis incidence are of 240 cases per 100 000 in the United States. The human and economic burden of sepsis is considerable (Grossi et al., 2006, Surg. Infect. (Larchmt), 7:S87-S91). Despite advances in infectious diseases and critical care management and numerous attempts to develop new treatments, sepsis mortality rate remains unacceptably high, ranging from 20% to 50%. Recognizing the signs of severe blood infections and/or severe sepsis and making an early and accurate diagnosis of it are the key to improving care and increasing the survival rate. Indeed, rapid diagnostics could increase patient survival by reducing the time interval between blood sampling and antimicrobial therapy application.
A need exists for efficient and accurate diagnostic tests for bodily fluid infections that would i) recover sufficient microbial cells for their detection, ii) recover microbial cells rapidly, iii) recover a large diversity of microbial species and iv) identify pathogens rapidly and accurately. The present invention seeks to meet these and other needs.
For several decades, different strategies have been used to try to meet such needs. The current standard is a broth-based blood culture system that favors growth of a majority of microorganisms present in a blood sample allowing them to multiply to a detectable level (Cockerill et al., 1996, J. Clin. Microbiol., 34:20-24; Murray et al., 1991, J. Clin. Microbiol., 29:901-905). However, such technique involves subsequent subculture on solid media for isolation and identification of microbial species. Consequently, several days are required to obtain an accurate diagnosis.
Saponins are naturally occurring surface-active glycosides having surfactant properties. They are mainly produced by plants but also by lower marine animals and some bacteria. They consist of a sugar moiety linked to a hydrophobic aglycone (sapogenin). The great complexity of saponin's structure arises from the variability of the aglycone structure, the nature of the side chains and the position of attachment of these moieties on the aglycone (Francis et al., 2002, British J of Nutrition, 88: 587-605). Saponins are known to interact with membranes of eukaryotic cells. Saponins are commonly used at 0.04%-0.2% concentrations to permeabilize plasma membranes. Such permeation may even lead to the destruction of the membrane with succeeding cell death. This process is function of the applied concentration and specific molecular structure of the saponin used (Melzig, et al. 2001, Planta Med., 67:43-48). It has been showed that the membrane binding site is cholesterol (Milgate et al. 1995, Nutrition Research, 15, no. 8; 1223-1249). Once bound to cholesterol, saponins induce changes in the membrane structure and permeability associated with disturbance of the ionic homeostasis between the intracellular and extracellular compartment. In yeast, molecules of ergosterol are found in the membrane instead of cholesterol. Studies showed that steroidal saponins (neutral saponins) preserve both hemolytic and antifungal activities, while triterpenoid saponins (acid saponins) show only hemolytic activity with no detectable antifungal activity. It is suggested that triterpenoid saponins may have a weaker affinity for ergosterol than cholesterol (Takechi et al. 2003, Phytother. Res., 17:83-85). Leconte et al. (Leconte et al. 1997, Phytochem., 44:575-579) demonstrated that cycloiridals, a class of triterpenoid from various Iris species, were able to stabilize yeast membranes after a disruption treatment by steroidal saponins. Triterpenoid saponins have been detected in many legumes such as soybeans, beans, peas and lucerne, as well as in alliums, tea, spinach, sugar beet, quinoa, liquorice, sunflower, horse chestnut and ginseng. One extensively studied group of triterpenoid saponins is produced from Quillaja saponaria, a tree native to the Andes region (Francis at al., 2002, British J of Nutrition, 88: 587-605). Saponins represent 20-25% of the extractable material from this source (Barr, et al., 1998, Ad Drug Deliv Rev, 32: 247-271). Commercially available saponin preparations may inhibit bacterial growth. Low molecular weight antibacterial contaminants may be removed from commercially available saponins by purification of the extracts by filtration (Dorn, G., Detoxification of saponins, U.S. Pat. No. 3,883,425, 1975).
Dorn et al. (U.S. Pat. No. 4,164,449) developed a method to lyse blood components with a minimum of 0.1 mg/mL and a maximum of 20 mg/mL of purified saponin. This method concentrates microbial cells by centrifugation and recovered cells are inoculated on an agar plate. A product based on this method is sold commercially as the Isostat®/Isolator™ (formerly named Isolator™ 10) and contains 1.83 mg/mL of purified saponin once mixed with the blood sample (Carter-Wallace, Inc., Cranbury, N.J. 08512-0181). This method allows detection of low-level bacteremia and fungemia caused by Enterobacteriacae, Staphylococcus epidermidis and yeasts within 1 to 2 days (McLaughlin et al. 1983, J. Clin. Microbiol., 18:1027-1031; Kiehn et al., 1983, J. Clin. Microbiol., 18:300-304). The increased sensitivity and shorter detection time may be due to the concentration of microbial cells from the initial blood sample volume. Another explanation for the improved detection obtained with Isolator™ 10 may be related to the release of intracellular microorganisms after lysis of some white blood cells by the saponin treatment. (Taylor, 1994, Eur. J. Clin. Microbiol. Infect. Dis., 13:249-252; Murray et al., 1991, J. Clin. Microbiol., 29:901-905). Some manufacturers of blood culture systems have supplemented their blood culture media with saponin (Murray et al., 1991, J. Clin. Microbiol., 29:901-905; Becton Dickinson BACTEC™ system; Hoffman La Roche biphasic Septi-Chek system).
Several groups compared blood culture media supplemented with saponin (varying from 0.03 mg/mL to 2 mg/mL of saponin when combined with a blood sample) or Isolator™ 10 product (1.83 mg/mL of saponin when combined with a blood sample) to the standard blood culture media to detect microorganisms in septicemic patients. These references suggest that microorganisms detection in blood specimen cannot be based only on a method using saponin. Indeed, they showed that Isolator™ 10 was not efficient for detection of Pseudomonas species in low-level bacteremia (Kiehn et al., 1983, J. Clin. Microbiol., 18:300-304; Henry et al., 1983, J. Clin. Microbiol., 17:864-869; Murray at al., 1991, J. Clin. Microbiol., 29:901-905). Another group found similar limitations for the detection of anaerobic species (McLaughlin at al., 1983, J. Clin. Microbiol., 18:1027-1031).
Spears et al. (EPO Publication No. 0,745,849) reported the use of a saponin or Triton™ in saline solution for whole blood lysis. Their method aims to process blood specimens in order to remove inhibitors of subsequent nucleic acids analysis. In this method, the blood sample is lysed by the addition of saponin to about 0.2 to 0.5% (2 to 5 mg/mL).
Another method, without saponin, was used to concentrate microorganisms from the initial blood sample volume (Bernhardt at al., 1991, J. Clin. Microbiol., 29:422-425). Blood sample is centrifuged to form density gradient with Ficoll™-hypaque to separate red blood cells from white blood cells. The upper layer containing white blood cells is filtered through a 0.22 μm pore size filter to retain microbial cells on the filter membrane. The filter is then placed on top of an agar plate to allow microbial growth. With this method, all microorganisms were detected within 18 hours after filtration in comparison to 24-48 hours with standard culture. However, among the 12 bacterial species tested in spiked blood sample, only the Pseudomonas aeruginosa spiked sample allowed for the recovery of microorganisms equivalent to blood culture on agar plate.
In brief, actual methods of detection remain time-consuming mainly due to the use of microbial cell culture to detect isolated pathogens. Furthermore, blood culture systems (e.g. BACTEC™, Isostat®/Isolator™) all use non-heated aqueous saponin solutions.
Saponins' structure may undergo chemical transformations during storage or processing which in turn may modify their properties and activity. The use of heated saponin derivative in hematology has been reported (EPO Publication No. EP 1,422,509). This method aims at red blood cells lysis with saponin while quenching the lysis activity to preserve white blood cells for further analysis. This saponin derivative solution (50 mg/mL), heated at 121° C. for 30 minutes, was used in combination with an acid and/or surfactant to allow a broader range of saponin concentrations (0.02-0.035 mg/mL when combined with a blood sample). Furthermore, this patent describes a heating procedure that enhances the stability of the reagents over time. HPLC analysis indicated that this heating process led to an additional unidentified peak, which further appeared to have no lytic capability. It was suggested that the heating procedure removed unstable components from saponin that could degrade over time. It has been reported that saponins from intact soybeans are hydrolyzed into Group B and E saponins upon heating in alkaline solutions in the presence of iron (Güçlü-Üstündag, Ö. et al., 2007, Crit. Rev Food Sci Nut: 231-258). Moreover, the heating procedure has been shown to modify biological functions of soy saponins (Okubo, K., et al, Oxygen-Saponins used in food and agriculture, Plenum Press, NY, 1996).
In the majority of the above described reports, saponin solutions are filtered purified after dissolution using 0.8 to 0.2 μm filtering devices with various types of membranes and keeping the filtrate. The effect of filtration in each case may be complex to measure since variations in the ability of saponin from different sources to form micelles around cholesterol molecules may be due to differences in molecular structures contained (San Martin et al., 2000, J. Sci. Food Agric., 80:2063-2068). Since Quillaja saponin is a biological extract rather than a synthetic compound, commercial products may contain various impurities such as salts, or surface-active molecules which affect micelle-forming capabilities of saponin molecules (Mitra et al., 1997, J. Agric. Food Chem., 45:1587-1595). These concentrations are subject to vary since the dissolution of saponin crude extracts in water is difficult to achieve efficiently. Quillaja bark saponin is soluble in alcohol, ether, acetone, ethyl acetate and/or glacial acetic acid (Güçlü-Üstündag, Ö., et al., 2007, Crit. Rev Food Sci Nut: 231-258).
In U.S. Pat. No. 3,883,425, an aqueous saponin solution is prepared by keeping the residue retained by the filtration device instead of the filtrate. This patent describes a procedure aiming at removing constituents in the saponin extract that have a molecular weight of less than about 600, described as being toxic to microbial organisms. During filtration, these toxic molecules pass through the membrane and remain in the filtrate.
Recent advances in molecular biology have allowed the development of tools for sensitive and accurate identification of bloodstream pathogens by bypassing microbial culture steps. Progress in nucleic acid amplification technologies allowed advances in the detection of small amounts of nucleic acids. However, new challenges are associated with these technologies. A first challenge involves a need for the recovery of most microbial cells from a sample to detect microbial nucleic acids by amplification without any cell replication step, even when blood samples contain less than 10 CFU/mL. A second challenge involves the need for decrement of human genomic DNA/microbial genomic DNA ratios to favor microbial DNA amplification. A third challenge involves the need to control nucleic acid amplification inhibitors originating from blood (e.g. inhibitors of polymerase chain reaction (PCR). The present invention seeks to meet these and other needs. PCR is by far the most popular nucleic acid amplification technology. PCR-based diagnosis of microbial infections and genetic diseases may be reduced or blocked by the presence of PCR-inhibitory substances in blood samples (Hoorfar et al., 2004, J. Appl. Microbiol., 96:221-222). PCR inhibitors have been identified as mainly heme and leukocyte DNA, but also anticoagulants like EDTA and heparin. More recently, Immunoglobulin G in human plasma, hemoglobin and lactoferrin in erythrocytes and leukocytes respectively, also proved to be major inhibitors of diagnostic PCR from blood (Al-Soud et al., 2000, J. Clin. Microbiol., 39:485-493). A need exists for improving isolation of microorganism from blood specimen that may be applicable for detection of both bacteria and fungi.
Among published and commercially available products, some methods involve a total simultaneous lysis of red and white blood cells as well as microbial cells to purify total nucleic acids afterwards (Jordan et al, 2005, J. Mol. Diagn., 7:575-581; NucliSens® easyMAG™ system from BioMérieux; SeptiFast prep kit from Roche Diagnostics; and Isoquick® nucleic acid extraction kit from ISC BioExpress). A disadvantage of this strategy is the presence of large amounts of blood cells nucleic acids as compared to microbial cells nucleic acids. This may prevent a good analytical sensitivity of microbial nucleic acids detection.
Other methods proceed to lysis of blood and microbial cells in separate steps followed by purification of nucleic acids. With this strategy, some groups use a hypotonic shock to lyse red blood cells and a combination of 0.2% SDS-proteinase K to lyse white blood cells before lysing yeast cells with an enzymatic digestion (White et al., 2006, Clin. Infect. Dis., 42:479-486; Loeffler et al., 2002, J. Clin. Microbiol., 40:2240-2243). Another group uses the Isolator™ 10 technology to lyse blood cells. The harvested yeast cells mixed with blood cells residues are then enzymatically digested and nucleic acids are purified (U.S. Pat. No. 5,645,992). These methods were developed to only detect fungal species.
The MolYsis Basic5 kit from Molzym uses guanidium thiocyanate and a chaotropic-resistant DNase to lyse blood cells and remove their nucleic acids prior to bacterial cell lysis and nucleic acid extraction. Bougnoux et al. (Bougnoux et al., 1999, J. Clin. Microbiol., 37:925-930) use a combination of sucrose and Triton™ X-100 to treat blood samples spiked with Candida cells to lyse blood cells. After centrifugation of unlysed cells, the pellet is resuspended and digested with DNasel to degrade nucleic acids released from white blood cells. After digestion, the cell suspension including the spiked Candida cells is centrifuged. The supernatant is discarded and the pellet is resuspended and submitted to a lyticase treatment to digest the yeast cell walls prior to their nucleic acid extraction.
The SeptiFast prep kit (LightCycler® SeptiFast Test MGRADE) was developed for the detection of both bacteria (19 different groups and 25 different species) and fungi (6 different species). The analytical sensitivity of this test is approximately 30 CFU of microbe/mL of blood. This system requires numerous handling steps and takes about 2 hours of handling prior to the extraction of human and microbial genomic DNA. Furthermore, it was shown that a majority of blood samples collected from septicemic patients may contain as low as 10 colony-forming units (CFU) of microbes per mL of blood (Jonsson et al., 1993, APMIS, 101:595-601).
In addition to nucleic acid-based methods, detection and/or identification of microbes may be performed by detecting phenotypical characteristics, microbial antigens, cellular components and/or physiological activities of microbial cells. Multiparametric analysis of microbial markers is useful for identifying microbes, for example, using microanalytical methods and microfabricated devices (Link et al., 2007, Nat. Rev. Microbiol. 5:680-688; Weibel et al., 2007, Nat. Rev. Microbiol. 5:209-218). Viable microbial cells (and/or metabolically active microbes) may be required to perform such analysis (Metzger, S. et al, ASM general meeting 2008, Abstract C-145; Metzger, S. et al, ASM general meeting 2008, Abstract C-005)
The present description refers to a number of documents, the content of which is herein incorporated by reference in their entirety.